| Backgroud and ObjectivesAstragali radix, a well-known traditional Chinese herbal medicine, is the essential ingredient in more than 200 Chinese herb formulas. Pharmacological studies and clinical practices indicate that Astragali radix possesses various biological functions, such as hepatoprotective properties, antioxidative effects, immunostimulating effects, and induces vasodilatation. More than 100 chemical constituents of Astragali radix have been isolated and identified to date. Among them, isoflavonoids is the main beneficial compounds responsible for its pharmacological activities and therapeutic efficacy.The therapeutic mechanism of traditional Chinese herbs is complicated owing to the multiple and complex active compounds. The pharmacokinetic characteristics of the active ingredients in herbs are useful to predict both the efficacy and the potential toxicity of a medicine as well as to optimize dose regimens and avoid adverse effects. Therefore, pharmacokinetic study of the main bioactive components of TCM is important for clinical applications and may help clarify their pharmacological action. To date, several analytical methods, including LC-MS/MS, which is being more and more widely applied in pharmaceutical research, have been used to quantify the marked compounds in biological samples of animals following oral administration of Chinese herb formulas including Astragali radix and the ethanol extract of Astragali radix. However, these methods have shortcomings more or less, and the water extract of Astragali radix was not studied in these studies. In addition, isoflavonoids, the main constituents of Astragali radix, belongs to flavonoid compounds, and contains free hydroxyl groups, which can be easily conjugated by UDP-glucuronosyltransferases (UGTs) forming the glucuronides in liver and intestine. The real active compounds of Astragali radix may be the combination of absorptive constituents and their metabolites in vivo, and many glucuronides of flavonoids showed several pharmacological activities. Therefore, the pharmacokinetic study of these glucuronides will promote the study of the pharmacological activities of Astragali radix.On the other hand, expect metabolism, absorption, hydrolysis and excretion in intestine and intestinal recycling are proposed to be involved in the disposition of flavonoids, which impacts their systemic and local bioavailability. It was stated that aglycone of flavonoids got into the cells through free diffusion. However, glucosides, with high polarity, cannot cross the cell monolayer until they are hydrolyzed by lactase-phlorizin hydrolase (LPH). Besides, some glucosides can be transported into enterocytes via sodium-dependent glucose transporter 1 (SGLT-1) and hydrolyzed by β-glucosides hydrolases in enterocytes. The glucuronides of flavonoids possess higher polarity and can be excreted by efflux transporters including breast cancer resistance protein (BCRP) and multidrug resistance proteins (MRPs). The bioavailability barrier consisting efflux transporters drug metabolizing enzymes could hinder the development of flavonoids in to drugs.Intestinal recycling is a disposition mechanism of flavonoids in liver and intestine. Enterohepatic recycling involves the action of liver to excrete glucuronides and action of bacterial β-glucuronidase to release the aglycones, which could enter the enterocytes and form glucuronides to complete the enterohepatic recycling. Enteric recycling refers to the process that glucuronides excreted by the enterocytes, are hydrolyzed to aglycones by bacterial β-glucuronidase for reabsorption in colon, completing the enteric recycling. Local recycling refers to the process that glucuronides excreted by the enterocytes, are hydrolyzed to aglycones by enterocyte-derived β-glucuronidase, and the aglycones could be reabsorbed in the small intestine, completing the local recycling. These three recycling could prolong the half-lives and retention time of flavonoids, increasing the enterohepatic bioavailability, which would finally to enhance the pharmacological activities of liver and intestine.Calycosin-7-O-β-glucoside (CG), an isoflavonoids glucoside, is the most active constituent in Astragali radix and has been reported to have anti-inflammation, antioxidative and neuroprotective effects. The bioavailability of CG and its aglycone, calycosin, is reported to be low. However, few studies have been reported on the pharmacokinetics and the absorption and disposition process of CG and calycosin.Therefore, the objectives of this study are to investigate the pharmacokinetic characteristics of water extract of Astragali radix and CG, and the mechanism of absorption, hydrolyzation and disposition of CG. This study would provide the theoretical foundation for the pharmacological studies of Astragali radix.Methods1. Study of pharmacokinetic profiles of the water extract of Astragali radix and CGThe rat pharmacokinetic model was used for the study pharmacokinetics of isoflavonoids and their metabolites in rat plasma following oral administration of the water extract of Astragali radix and CG and its metabolites in rat plasma following oral, intravenous and intraperitoneal administration of CG. The blood concentration was detected by UHPLC-MS/MS. Through the drug-time curve, to investigate the pharmacokinetic profiles and characteristics of compounds in blood and judge hydrolyzed organ of CG.2. Study of characteristics of absorption and hydrolysis of CG in intestineAn in situ rat intestinal perfused model and a series of hydrolysis experiments were used for the study of absorption and hydrolysis of CG. The effects of LPH inhibitor and glucose transporters inhibitors on the disposition of CG were investigated to figure out whether CG could simultaneously participate in enterohepatic, enteric and local recycling.3. The effects of UGT and transporters on the disposition of CGIn vitro glucuronidation incubation via human liver microsomes (HLMs), human intestina microsomes (HIMs), rat liver microsomes (RLMs), rat intestinal microsomes (RIMs) and human UGT isoforms were investigated for the glucuornidation characteristics of CG and calycosin. In addition, enzyme kinetics of HLMs, HIMs, RLMs, RIMs and the main UGT isoforms were conducted. The Caco-2 cell line and knockout mice were also used to determine the efflux transporters involved in the transport of calycosin.Results1. Study of pharmacokinetic profiles of the water extract of Astragali radixA validated LC-MS/MS method was applied to the determination of the plasma concentrations of eight constituents after oral administration of water extract of Astragali radix. Isoflavonoids and their metabolites are the major type of constituents absorbed in plasma, including CG and CG glucuronide (CG-G’), calycosin glucuronide (C-G’), formononetin and its glucuronide (F-G’) and daidzein glucuronide (D-G’), and their plasma concentrations were much higher than their parents. Among them, Cmax of C-G’ was highest (more than 1 μ.g/mL). Daidzein was not found in the water extract of Astragali radix, but D-G’ could be detected in the plasma. It was speculated that daidzein could be generated from other isoflavonoids through demethylation and dehydroxylation and was glucuronidated to D-G’ in the enterocytes. The phenomenon illustrated the biotransformation between these isoflavonoids was extensively existence in the body. The constituents detected in this study achieved their maximum plasma concentrations within 1 h, demonstrating rapid absorption from the gastrointestinal tract. And glucuronides had longer half-life time.2. Study of pharmacokinetic profiles of CGThe pharmacokinetic characteristics of CG and its metabolites were studied after intravenous (0.5 mg/kg), intraperitoneal (10 mg/kg), and oral (10 mg/kg) administrations of CG. After oral administration of CG, the Cmax and AUC0-∞ values of C-G’ were 1189.66±346.95 ng/mL and 111.16±34.18 min·mg/mL, respectively, which were much higher than those of CG glucuronide and CG. After intraperitoneal administration, CG exhibited the highest Cmax (965.24±133.53 ng/mL). However, the AUC0-∞ of CG (72.64±22.08 min-mg/mL) was not the highest because the elimination of CG was very fast (t1/2=77.5±17.91 min), and its plasma concentration was lower than 10 ng/mL after 4 hours. The AUC0-∞ of C-G’ was 99.11±45.90 min·mg/mL. After intravenous administration of CG, the plasma concentrations of CG glucuronide and C-G’ were considerably low, and the AUC0-∞ of CG was 5.39±1.02 min·mg/mL. Thus, the absolute oral bioavailability of CG was 0.304%. This indicated that the intestinal disposition would significantly affect the pharmacokinetics of CG.3. Study of characteristics of absorption and hydrolyzation of CG in intestineCG was perfused in an in situ rat perfused intestinal model. The results indicated that expect for CG, the aglycone calycosin and a large amount of C-G’were found in the perfusate. In addition, C-G’ could be detected in the bile. CG was rapidly absorbed in the duodenum, jejunum, and ileum, with rates higher than those in the colon. Moreover, permeability of 2.5 μM in all intestinal segments were higher than those of 10 mM, which indicated that transporters could be involved in the absorption of CG. The absorption of CG in the duodenum, jejunum, and ileum reached 17% while only 6% in colon. The metabolism of calycosin glucuronide in jejunum was approximately 4% while nearly zero in colon.CG was co-perfused with 20 and 40 mM LPH inhibitor glucurolactone, the absorption of CG and the excretion of C-G’were not changed, and the concentrations of C-G’and CG-G’in bile and plasma were also not changed, indicated that CG was not hydrolyzed by LPH. This result was validated by using daidzin as the positive control. On the other hand, CG was co-perfused with glucose, the absorption of CG and the excretion of C-G’were reduced significantly, and the concentrations of C-G’ and CG-G’in bile and plasma were also reduced. Because glucose could saturate the glucose transporters, the results indicated that the absorption of CG was transported by glucose transporters.CG was co-perfused with 100,200 and 400 μM sodium-dependent glucose transporter 1 (SGLT-1) inhibitor phloridzin, the absorption of CG and the excretion of C-G’were reduced in a dose dependent manner, and the concentrations of C-G’and CG-G’in bile and plasma were also reduced, which indicated that the absorption of CG was transported by SGLT-1. However, phloridzin played no role on CG absorption in the colon, as evidenced by the absence of SGLT-1 expression in the rat colon. CG was co-perfused with 50,100 and 200 μM glucose transporter 2 (GLUT-2) inhibitor phloretin, the absorption of CG and the excretion of C-G’were not changed, and the concentrations of C-G’ and CG-G’ in bile and plasma were also not changed, indicated that CG was not transported by GLUT-2.CG was co-perfused with ko143 (50 μM), the inhibitor of BCRP, and MK571 (50 μM), the inhibitor of MRP2, the absorption of CG was not changed, but the excretion of C-G’ was significantly reduced in all four segments of the intestine, which indicated C-G’ was excreted by BCRP and MRP2. In addition, the rat perfused intestinal model demonstrated that CG was not hydrolyzed by LPH, but the concentration of C-G’ in perfusate, bile and plasma was highest, indicating that the hydrolytic site of CG is likely to be inside the enterocytes.25,50 and 100 μM CG and intestinal S9 were incubated with or without 2.5,5 and 10 mM BSβG inhibitor gluconolactone and 1.25,2.5 and 5 mM glucocerebrosidase inhibitor conduritol B epoxide. The results showed that these inhibitors could significantly inhibit calycosin formation in a dose dependent manner, indicating CG was mainly hydrolyzed in the enterocytes after transported by SGLT-1.When incubated with β-glucuronidase from Escherichia coli, the concentration of calycosin glucuronides was reduced in the perfusate and bile. This indicated that calycosin glucuronide could be hydrolyzed into calycosin by bacterial β-glucuronidase in colon, which could be absorbed into enterocytes and metabolized, participated in enterohepatic and enteric recycling. However, when calycosin glucuronide was incubated in blank perfusate, the concentration was not changed. This indicated that calycosin glcuuronide could not be hydrolyzed into aglycone by enterocyte-derived β-glucuronidase and local recycling was not involved in the intestinal disposition of CG.4. The effects of UGT and transporters on the disposition of CGA quadrupole-time of flight (Q-TOF) tandem mass spectrometer was used to determine the molecular weight and structure of the metabolites. For CG and calycosin, one or two metabolites were formed after incubation with microsomes. The pseudo-molecule ions and fragment ions corresponed to the mono-gluccuronides, indicating that the mono-glucuronide was generated via glucuronidation incubation. 12 expressed human UGT isoforms were used to incubated with CG and calycosin, the result showed that UGT1A10 was the major isoform for CG, UGT1A9 was the major isoform for 3’-OH of calycosin and UGT1A1 was the major isoform for 7-OH of calycosin. The enzyme kinetics of CG and calycosin in HLMs, HIMs, RLMs and RIMs were difference, indicating that the metabolism of CG and calycosin demonstrated species difference.In the transport study of Caco-2 cell line, calycosin could be metabolized into two glucuronides and then be excreted. BCRP inhibitor and MRPs inhibitor significantly reduced the efflux rate and Fmet of calycosin glucuronides in the Caco-2 cell line while significantly enhanced the intracellular amount of calycosin glucuronides. The results suggested that BCRP, MRP1/2/3 might be efflux transporter for calycosin glucuronides.In addition, the pharmacokinetic study of CG in Fvb wild mice, Fvb BCRP-/-mice, Fvb MRP2-/- mice, Fvb MDR1-/- mice, and Fvb MRP1-/- mice. The results showed that Cmax and AUC of calycosin glucuronide in Fvb BCRP-/- mice and Fvb MRP2-/- mice were significantly increased, but Cmax and AUC of calycosin glucuronide in Fvb MDR1-/- mice and Fvb MRP1-/- mice were not changed. The results indicated that the transporter in the basolateral side of Caco-2 cell line for calycosin glucuronide could be MRP3.Conclusions1. This is the first report on pharmacokinetic studies of the glucuronides of calycosin, CG, formononetin, and daidzein in vivo following the oral administration of water extract of Astragali radix. The higher plasma concentration of the glucuronides and the biotransformation between the bioactive isoflavonoids and their glucuronides suggested that, in addition to the parent compounds, we should focus on the metabolites and biotransformation between the components when conducting a drug efficacy study.2. CG could be subjected to enterohepatic recycling and enteric recycling, which were consisted by glucose transporters, hydrolase, UGT enzymes, and multiple efflux tranporters. After oral administration, CG would be transported by SGLT-1 into enterocytes and hydrolyzed by BSβG and glucocerebrosidase into calycosin in the upper small intestine. Calycosin will be metabolized into glucuronides in both hepatocytes and enterocytes, which were then rapidly excreted into bile and lumen. Glucuronides can be hydrolyzed back to its aglycone forms by bacteria-derived P-glucuronidase in the colon. The released aglycone, calycosin would be subsequently reabsorbed in the colon for enterohepatic and enteric recycling, completing the recycling process. Our findings contribute to improved understanding of the absorption and disposition of flavonoid compounds, which is essential for determining their potential as chemopreventive agents for LPH-deficient individuals. |
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